Anormalidades miocárdicas destacam-se entre as manifestações cardiovasculares da doença renal policística autossômica dominante (DRPAD). Para investigar a patogênese dessas manifestações, analisamos o fenótipo cardíaco em camundongos com diferentes perfis de deficiência de Pkd1. Avaliamos o modelo Pkd1cond/cond:Nestincre (CI), com cistos renais e hipertensão, na idade de 20-24 semanas, e heterozigotos para mutação nula em Pkd1 (Pkd1+/-; HT) entre 10-13 semanas, representando um modelo não cístico de haploinsuficiência gênica. Animais Pkd1cond/cond (não cístico; NC) e Pkd1+/+ (selvagem, SV) foram usados como controles. Análises ecocardiográficas de camundongos CI e HT revelaram diminuição da fração de ejeção do ventrículo esquerdo, indicando disfunção sistólica. A relação E/A e o tempo de desaceleração foram consistentes com disfunção diastólica em animais CI. Ecocardiografia por speckle-tracking mostrou redução na deformidade cardíaca (strain) nos modelos CI e HT. Os corações de ambos os grupos apresentaram índices de apoptose maiores e fibrose discreta. Neste cenário, investigamos galectina-3 (Gal-3) como modificador potencial do fenótipo cardíaco na DRPAD. Duplos-mutantes Pkd1cond/cond:Nestincre;Lgals3-/- (CIG-) e Pkd1+/- ;Lgals3-/- (HTG-) cursaram com melhora da função sistólica e de strain comparados a CIs e HTs, não diferindo de NCs e SVs. Animais HTG- apresentaram melhora parcial da função diastólica. Apoptose e fibrose cardíaca mostraram-se reduzidas em CIG-s e HTG-s, alcançando valores similares a NCs e SVs. Análises de western blot revelaram expressão de Gal-3 maior em corações CIs que NCs, porém o mesmo não ocorreu entre HTs e SVs. Os duplos-mutantes não apresentaram diferença na ureia sérica quando comparados a CIs e HTs, assim como nas frações de excreção de Na+, Cl- e K+. Por fim, empregamos um modelo renal cístico grave, homozigoto para um alelo que impede a clivagem da policistina-1 no sítio GPS (Pkd1V/V; VV), e mostramos que a ausência de galectina-3 aumentou a sobrevida em animais Pkd1V/V;Lgals3-/- (VVG-). Nossos resultados demonstram disfunção e alterações de deformidade miocárdica em diferentes modelos de deficiência de Pkd1, à semelhança da DRPAD humana, e revelam que knockout de Gal-3 resgata significativamente este fenótipo / Myocardial abnormalities stand out among ADPKD cardiovascular manifestations. To elucidate their pathogenesis, we analyzed the cardiac phenotype in distinct models of Pkd1-deficiency. We evaluated Pkd1cond/cond:Nestincre (CY) cystic, hypertensive mice at 20-24 weeks of age, and Pkd1+/- (HT) noncystic mice at 10-13 weeks, a model of gene haploinsufficiency. Pkd1cond/cond (noncystic; NC) and Pkd1+/+ (wild type, WT) animals were used as controls. Echocardiographic analyses in CY and HT mice revealed decreased left ventricle ejection fraction (LVEF), indicating systolic dysfunction, as well as E/A ratios and deceleration times consistent with diastolic dysfunction in CY animals. Speckle-tracking echocardiography showed reduced cardiac deformability in both models. CY and HT hearts presented higher apoptotic rates and mild fibrosis. In this scenario, we investigated galectin-3 (Gal-3) as a potential modifier of the ADPKD cardiac phenotype. Double mutants Pkd1cond/cond:Nestincre;Lgals3-/- (CYG-) and Pkd1+/-;Lgals3-/- (HTG-) displayed improved systolic and deformability parameters compared to single mutants, while such values did not differ from NCs and WTs. HTG-s presented a partial improvement in diastolic function. CYG- and HTG- hearts showed decreased apoptosis and fibrosis, reaching NC and WT baselines. Western blot analyses revealed higher Gal-3 expression in CY than NC hearts but no difference between HT and WT mice. CYG- and HTG- animals showed no difference in BUN and in the fractional excretion of Na+, Cl- and K+ compared to CYs and HTs. We also employed a more severe renal cystic model, homozygous for an allele that hinders polycystin-1 cleavage at the GPS site (Pkd1V/V; VV), and showed that Pkd1V/V;Lgals3-/- mice present longer survival than VVs. Our findings demonstrate myocardial dysfunction and abnormal deformability in different Pkd1-deficient models, reproducing human ADPKD, and reveals that Gal-3 knockout significantly rescues this phenotype

Vários estudos mostram que na doença renal policística autossômica dominante os cistos surgem a partir de um mecanismo de "dois-golpes". A patogênese das manifestações não-císticas, contudo, é pouco compreendida. Neste estudo usamos uma linhagem de camundongos endogâmica com uma mutação nula em Pkd1, onde animais heterozigotos apresentam formação cística renal mínima até 40 semanas de idade. O clearance de inulina e o número de glomérulos foram menores em machos Pkd1+/- que Pkd1+/+, enquanto o volume glomerular médio foi maior em heterozigotos. A excreção urinária de NO2/NO3 não diferiu significantemente entre os dois grupos. Avaliamos a osmolalidade urinária máxima em machos e fêmeas Pkd1+/- and Pkd1+/+, porém não foi detectada diferença significante entre os grupos heterozigoto e selvagem. Nossos resultados oferecem evidência direta de que a haploinsuficiência de Pkd1 resulta em anormalidades anatômicas e funcionais renais e sugerem que o estado haploinsuficiente de Pkd1 possa resultar na redução do número de néfrons por diminuir a ramificação tubular renal durante a nefrogênese / Several studies show that in autosomal dominant polycystic kidney disease cysts arise through a "two-hit" mechanism. The pathogenesis of non-cystic features, however, is poorly understood. In this study we used an inbred mouse line with a null mutation of Pkd1, where heterozygotes had minimal renal cyst formation up to 40 weeks of age. Inulin clearance and the number of glomeruli were lower in Pkd1+/- than in Pkd1+/+ males, while a higher average glomerular volume was observed in heterozygotes. The urinary excretion of NO2/NO3 did not significantly differ between the two groups. Maximal urinary osmolality was evaluated in Pkd1+/- and Pkd1+/+ males and females, but no significant difference was detected between the heterozygous and the wild type groups. Our results provide direct evidence that haploinsufficiency for Pkd1 results in anatomic and functional abnormalities of the kidney and suggest that Pkd1 haploinsufficiency may result in a reduced number of nephrons by diminishing renal tubule branching during nephrogenesis

A maior parte dos casos de doença renal policística autossômica dominante (DRPAD) é causada por mutações no gene PKD1 (Polycystic Kidney Disease 1). O insulto por isquemia/reperfusão (IR) constitui-se em uma causa freqüente de lesão renal aguda, incluindo a população de pacientes com DRPAD, mas a relação entre policistina-1 e IR é essencialmente desconhecida. Uma vez que a policistina-1 modula proliferação, diferenciação celular e apoptose em sistemas de cultura de células, sua menor atividade biológica na DRPAD poderia favorecer um maior grau de lesão renal. Utilizamos uma linhagem endogâmica de camundongos 129Sv com uma mutação nula em Pkd1 para testar esta hipótese. Camundongos Pkd1+/- não apresentam cistos renais até 12 semanas de vida, constituindo-se em um modelo puro de haploinsuficiência para este gene. Um insulto IR bilateral de 32 min foi induzido em camundongos machos de 10-12 semanas de idade, heterozigotos e selvagens, por meio do clampeamento reversível de ambos os pedículos renais. Os animais foram analisados 48 h, 7 dias (d) e 14 d após o insulto. Camundongos Pkd1+/- apresentaram FENa, FEK e SCr mais elevadas que animais Pkd1+/+ 48 h após IR. O dano cortical residual foi mais severo em heterozigotos que em selvagens em todos os tempos avaliados. A marcação para PCNA também foi mais alta em camundongos Pkd1+/- que Pkd1+/+ 48 h e 7 d pós-IR, enquanto a taxa de apoptose e a infiltração inflamatória intersticial foram maiores em heterozigotos que em selvagens nos seguimentos de 48 h, 7 d e 14 d pós-IR. A expressão renal de p21 foi menor nos camundongos Pkd1+/- que Pkd1+/+ no tempo de 48 h pós-insulto, tanto no nível transcricional como traducional. Análises adicionais realizadas 6 semanas após o insulto IR revelaram dilatação tubular e formação de microcistos nos camundongos haploinsuficientes para Pkd1, assim como fibrose renal aumentada nesses animais, comparados aos camundongos selvagens. Por fim, um insulto de 35 min de isquemia/reperfusão acompanhou-se de uma mortalidade precoce substancialmente maior nos animais Pkd1+/-. Esses achados sugerem que isquemia/reperfusão induza uma lesão mais severa em rins de camundongos haploinsuficientes para Pkd1, um processo aparentemente dependente de uma deficiência relativa da atividade de p21, assim como dilatação tubular e formação de microcistos. Em conjunto, nossos resultados sugerem que a heterozigose para mutação nula em Pkd1 em camundongo (e talvez em humanos) esteja associada a um risco aumentado para lesão renal por isquemia/reperfusão e a um pior impacto desse insulto sobre a progressão da doença renal. / The majority of autosomal dominant polycystic kidney disease (ADPKD) cases are caused by mutations in the PKD1 gene. Ischemia/reperfusion is a frequent cause of acute kidney injury, including the ADPKD patient population, but the relationship between polycystin-1 and ischemia/reperfusion is essentially unknown. Since polycystin-1 modulates cell proliferation, cell differentiation and apoptosis in cell culture systems, its lower biological activity in ADPKD might amplify the degree of renal injury. Using an inbred 129Sv mouse line with a Pkd1-null mutation, 32-min renal ischemia/reperfusion was induced in 10-12 week-old male non-cystic mice, heterozygotes and wild types. The animals were analyzed at 48h, 7 days (d) and 14d after the insult. Pkd1+/- mice showed higher FENa, FEK and SCr than Pkd1+/+ animals at 48h of follow-up. The residual cortical damage was more severe in heterozygotes than wild types at all evaluated time points. The PCNA staining was also higher in Pkd1+/- than Pkd1+/+ mice at 48h and 7d, while cell apoptotic rates and the interstitial inflammatory infiltration were higher in heterozygotes than wild types at 48h, 7d and 14d postischemia/ reperfusion. The expression of p21 was lower in Pkd1+/- than Pkd1+/+ kidneys at 48h, both at the transcriptional and translational levels. Additional analyses performed 6 weeks after the insult showed tubular dilatation and microcyst formation in the haploinsufficient mice, and increased renal fibrosis in these animals compared to wild types. Thirty-fivemin ischemia/reperfusion, at last, was accompanied by a substantially higher early mortality of Pkd1+/- animals. These findings suggest that ischemia/reperfusion induces a more severe injury in kidneys of Pkd1- haploinsufficient mice, a process that is apparently dependent on a relative deficiency of p21 activity, as well as tubular dilatation and microcyst formation. Altogether, our results suggest that mouse Pkd1-null heterozygosity (and maybe human) is associated with a higher risk for renal ischemia/reperfusion injury and with a worse impact of this insult upon renal disease progression.

Les maladies rénales chroniques sont devenues un enjeu majeur de santé publique. Qu’elle qu’en soit la cause initiale, la MRC est caractérisée par une réduction néphronique progressive, aboutissant au remplacement des néphrons sains par un tissu fibreux et au déclin de la fonction rénale. Les mécanismes de progression de la MRC sont encore mal compris, mais il a été suggéré que le développement des lésions tubulo-interstitielles joue un rôle essentiel dans le déclin de la fonction rénale. Deux éléments physiopathologiques cruciaux dans le développement de ces lésions sont représentés par (i) l’activation de la voie du récepteur à l’EGF (epidermal growth factor) (EGFR), et (ii) la protéinurie et ses conséquences pour les cellules tubulaires. Les médiateurs communs à ces deux phénomènes ne sont pas connus. Mon travail de thèse a consisté à caractériser une protéine commune à ces deux voies d’activation, ie la lipocaline2 (Lcn2), petite protéine de transport de fer, en étudiant ses voies d'activation et ses conséquences physiopathologiques. Nous montrons que le rôle pathologique de la voie de l’EGFR est gouverné par la surexpression de Lcn2. En effet, dans le contexte de réduction néphronique chirurgicale, (i) les animaux invalidés pour Lcn2 sont protégés du développement des lésions, et (ii) les souris exprimant un dominant négatif de l’EGFR dans le tubule rénal présentent une diminution de l’expression de Lcn2. Nous montrons également que l’invalidation de Lcn2 permet de ralentir la progression de la MRC dans un modèle de polykystose rénale dépendante de l’EGFR, les souris jck (juvenile cystic kidney). Parallèlement, nous montrons que la protéinurie induit également l’expression de Lcn2 dans les cellules tubulaires rénales dans différents modèles expérimentaux. De plus, nous montrons le rôle majeur de Lcn2 dans la progression de la MRC protéinurique, l’invalidation de Lcn2 limitant le développement des lésions rénales et la mortalité des animaux protéinuriques. Si le rôle délétère de Lcn2 est démontré dans différents modèles de néphropathie chronique, nous montrons que les voies moléculaires impliquées dans l’activation de Lcn2 et le rôle de cette protéine dépendent du contexte cellulaire. Nous prouvons que Lcn2 est un médiateur de l'effet mitogénique de l'EGFR, phénomène essentiel de la progression de la MRC, et nous montrons que l’activation de Lcn2 via l’EGFR est dépendante du facteur HIF1α. Cependant, nous démontrons également que l'expression de Lcn2 dans le contexte de protéinurie est dépendante du facteur ATF4 activé par le stress du reticulum endoplasmique (ER), et que Lcn2 est un médiateur de l'apoptose dépendante du stress de l'ER. Enfin, nous prouvons que l’inhibition pharmacologique du stress de l'ER permet une réduction de l’expression de Lcn2 dans les cellules tubulaires, et surtout, un ralentissement du déclin de la fonction rénale des animaux protéinuriques. Nous démontrons également l’importance de ces résultats chez les patients atteints de MRC. Nous identifions NGAL, l'analogue humain de Lcn2, comme un biomarqueur de progression dans la polykystose rénale dominante, et nous montrons qu’elle est fortement surexprimée dans le tissu rénal de patients protéinuriques. L’ensemble de ce travail permet de montrer que Lcn2 est un nouveau médiateur essentiel de multiples néphropathies chroniques. Lcn2 est impliquée dans l’effet mitogénique de l’EGFR ou la réponse apoptotique associée à la protéinurie durant la MRC. Nous ouvrons également de nouvelles perspectives thérapeutiques avec l'utilisation d'inhibiteurs du stress de l'ER dans les néphropathies protéinuriques humaines / Chronic kidney disease (CKD) is now a major public health concern. Whatever the initial kidney injury, CKD is characterized by progressive nephron reduction and kidney function decline. Tubulointerstitial lesions are an essential component of CKD progression, and are mediated by two crucial pathophysiologic elements: epidermal growth factor receptor (EGFR) activation, and proteinuria responsible of tubular cell damage. The aim of this study was to describe a common mediator of both these pathways, ie lipocalin2, an iron carrier protein, by identifying its activation pathways and its pathophysiologic consequences. We show the deleterious effects of the EGFR pathway during nephron reduction is mediated by the activation of Lcn2, which controls the mitogenic effect of EGFR. In fact, after nephron reduction, animals invalidated for Lcn2 are protected from lesions developpement. Moreover, a similar protective effect is seen in jck (juvenile cytic kidney) mice invalidated for Lcn2, a model of polycystic kidney disease EGFR-dependant. Otherwise, we show proteinuria induces Lcn2 expression in tubular cells of different experimental models, and Lcn2 invalidation slows lesion developpement and reduces mortality of proteinuric mice. We demonstrate that the Lcn2 role and activation pathways are dependant of these different models. We show Lcn2 is a mediator of the mitogenic effect of the EGFR, and Lcn2 activation is dependant of HIF1α stabilisation. However, we also show ATF4 is an activator of Lcn2 during endoplasmic reticulum (ER) stress induced by proteinuria in tubular cells. In this context, Lcn2 controls ER stress-induced apoptosis. Pharmacologic inhibition of ER stress in proteinuric animals decreases Lcn2 overexpression, and slows renal function decline. In patients suffering from CKD, we demonstrated NGAL (neutrophil gelatinase-associated lipocalin), the human analog of Lcn2, appears as a critical biomarker of autosomal dominant polycystic kidney disease progression. NGAL is also highly overexpressed in tubular cells in kidney biopsies of proteinuric patients. This work demonstrates Lcn2 is an essential mediator of multiple pathophysiologic components of CKD progression. Moreover, we open new therapeutic perspectives with the use ER stress modulators in proteinuric CKD

La Polykystose Rénale Autosomique Dominante (PKRAD) est une des pathologies héréditaires les plus fréquentes et affecte environ un individu sur 1000. Elle se caractérise par une importante variabilité clinique, notamment dans l’âge de survenue de l’insuffisance rénale terminale. Deux gènes sont en cause : le gène PKD1 situé sur le chromosome 16 (85% des cas) et le gène PKD2 situé sur le chromosome 4 (15% des cas). Les progrès majeurs dans la compréhension des mécanismes moléculaires impliqués ont permis le développement de stratégies thérapeutiques spécifiques, et de nouvelles questions surgissent : quels patients traiter ? Quand débuter les traitements ? La cohorte Genkyst, qui vise à inclure tous les patients suivis pour PKRAD dans la région Grand Ouest, nous a d’abord permis de décrire la variabilité génétique rencontrée dans la PKRAD. Nous avons ensuite démontré l’existence de fortes corrélations génotype-phénotype, en rapportant l’influence sur l’âge de survenue de l’insuffisance rénale terminale non seulement du gène en cause, mais aussi du type de mutation pour le gène PKD1. Enfin, l’analyse des données cliniques et génétiques de 1341 patients nous a permis de développer un algorithme pronostique, baptisé le PROPKD score, permettant de stratifier le risque de progression vers l’insuffisance rénale terminale. Nous espérons que ces travaux participeront à l’individualisation de la prise en charge des patients atteints de PKRAD, ce qui est un enjeu crucial à l’arrivée des nouveaux traitements. / Autosomal Dominant Polycystic Kidney Disease (ADPKD) is one of the most frequent Mendelian inherited disorders, and affects approximately one individual out of 1000. ADPKD is marked by a high clinical variability, especially regarding age at end-stage renal disease (ESRD). Two genes are identified: PKD1 located on the chromosome 16 (85% of the pedigrees) and PKD2 located on the chromosome 4 (15% of the pedigrees). Substantial progress in understanding the cellular mechanisms underlying ADPKD has triggered the development of targeted therapies, and new questions are arising: which patients should be treated? When should we begin these treatments? Thanks to Genkyst cohort, which aims to include all consenting ADPKD patients from the western part of France, we first described the important allelic variability encountered in ADPKD. Secondly, we demonstrated the important influence of not only the gene involved, but also of PKD1 mutation type. Last, the analysis of clinical and genetic characteristics of 1341 patients from the Genkyst cohort allowed us to develop a prognostic algorithm, named the PROPKD score for predicting renal outcome in ADPKD. Our hope is that these works will participate in the development of individualized medicine in ADPKD, which is crucial in the context of the emerging targeted therapies.

No description available.

Autosomal recessive polycystic kidney disease (ARPKD) is a rare severe inherited disease manifested by cystic renal disease, congenital hepatic fibrosis and dilatatation of bile ducts. The spectrum of clinical manifestations is very wide and variable, depends on the age at which the disease was manifested. In severe forms of the disease, it is possible to detect the first symptoms prenatally around the 20th week of pregnancy due to increased echogenic kidneys and the presence of oligohydramnios. The causal gene of this disease is thePKHD1 gene with protein product fibrocystin that is most likely contributing on maintaining the intracellular concentration of Ca2+ cations. The exact phatophysiology mechanism of ARPKD remains unknown. Phenotypic manifestations of this disease may overlap with mutations associated with other genes. One of the genes mimicking the ARPKD phenotype is the HNF1B gene. Mutations associated with HNF1B gene are the most common monogenic cause of developmental kidney abnormalities. HNF1B is a tissue-specific transcription factor that regulates the expression of PKHD1. In experimental part I worked on genetic analysis of the HNF1B gene in 28 patients who have not been confirmed ARPKD diagnosis by detection of 2 PKHD1 mutations. For the purposes of mutational screening, I used...